SsaM and SpiC interact and regulate secretion of Salmonella pathogenicity island 2 type III secretion system effectors and translocators

The type III secretion system (TTSS) encoded by Salmonella Pathogenicity Island 2 (SPI-2) is required for systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. The SPI-2 TTSS is activated after internalization of bacteria by host cells, and translocates effector proteins into and across the vacuolar membrane, where they interfere with several host cell functions. Here, we investigated the function of SsaM, a small protein encoded within SPI-2. An ssaM deletion mutant had virulence and intracellular replication defects comparable to those of a SPI-2 TTSS null mutant. Although the ssaM mutant was able to secrete the effector protein SseJ in vitro, it failed to translocate SseJ into host cells, and to secrete the translocon proteins SseB, SseC and SseD in vitro. This phenotype is similar to that of a strain carrying a mutation in the SPI-2 gene spiC, whose product is reported to be an effector involved in trafficking of the Salmonella vacuole in macrophages. Both ssaM and spiC mutants were found to oversecrete the SPI-2 effector proteins SseJ and PipB in vitro. Fractionation assays and immunofluorescence microscopy were used to investigate the localization of SsaM and SpiC in macrophages. No evidence for translocation of these proteins was obtained. The similar phenotypes of the ssaM and spiC mutants suggested that they might be involved in the same function. Pull-down and co-immune precipitation experiments showed that SpiC and SsaM interact within the bacterial cell. We propose that a complex involving SsaM and SpiC distinguishes between translocators and effector proteins, and controls their ordered secretion through the SPI-2 TTSS.     

SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins

Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.     

RtsA and RtsB coordinately regulate expression of the invasion and flagellar genes in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process. The bacterium responds by tightly regulating the expression of virulence genes. We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNA(PheU) in S. enterica serovar Typhimurium. RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein. In a random screen, we identified five RtsA-regulated fusions, all belonging to the Salmonella pathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells. We show that RtsA increases expression of the invasion genes by inducing hilA expression. RtsA also induces expression of hilD, hilC, and the invF operon. However, induction of hilA is independent of HilC and HilD and is mediated by direct binding of RtsA to the hilA promoter. The phenotype of an rtsA null mutation is similar to the phenotype of a hilC mutation, both of which decrease expression of SPI1 genes approximately twofold. We also show that RtsA can induce expression of a SPI1 TTSS effector, slrP, independent of any SPI1 regulatory protein. RtsB represses expression of the flagellar genes by binding to the flhDC promoter region. Repression of the positive activators flhDC decreases expression of the entire flagellar regulon. We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine.     

A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis.     

The Salmonella SpiC protein targets the mammalian Hook3 protein function to alter cellular trafficking

The Salmonella SpiC protein is secreted into the cytosol of macrophages via a unique type III secretion system that functions intracellularly to translocate proteins across the phagosomal membrane. The SpiC protein is required for survival within macrophages and inhibition of phagosome-lysosome fusion in vivo, and it is sufficient to inhibit endosome-endosome fusion in vitro. Here, we establish that SpiC targets the function of Hook3, a mammalian protein implicated in cellular trafficking. Purified GST-SpiC pulled down Hook3 from murine macrophages, and anti-Hook3 antibodies precipitated SpiC from the cytosol of Salmonella-infected macrophages. Expression of the spiC gene disrupted Golgi morphology in Vero cells and altered the distribution of lysosomes in macrophages, mimicking the phenotype of cells expressing a hook3 dominant-negative mutant. By inactivating Hook3 function, the SpiC protein may alter the lysosome network and prevent phagosome-lysosome fusion.     

SseA is required for translocation of Salmonella pathogenicity island-2 effectors into host cells

The Salmonella pathogenicity island-2 (SPI2) is a virulence locus on the bacterial chromosome required for intracellular proliferation and systemic infection in mice. Cell culture models and a murine model of systemic infection were used to address the role of an uncharacterized SPI2 open reading frame, designated as sseA, in Salmonella virulence. A Salmonella strain with an unmarked internal deletion of sseA displayed a phenotype that was similar to an SPI2-encoded type III secretion system apparatus mutant. Moreover, SseA was required for survival and replication within epithelial cells and macrophages. Murine infection studies confirmed that the DeltasseA strain was severely attenuated for virulence. Using immunofluorescence microscopy, the virulence defect in the DeltasseA strain was attributed to an inability to translocate SPI2 effector proteins into host cells. These data demonstrate that SseA is essential for SPI2-mediated translocation of effector proteins.     

Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.     

The invasion-associated type III secretion system of Salmonella enterica serovar Typhimurium is necessary for intracellular proliferation and vacuole biogenesis in epithelial cells

Type III secretion systems (TTSS) are used by Gram-negative pathogens to translocate proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) has two of these specialized systems, which are encoded on separate Salmonella pathogenicity islands (SPI-1 and SPI-2) and translocate unique sets of effectors. The specific roles of these systems in Salmonella pathogenesis remain undefined, although SPI-1 is required for bacterial invasion of epithelial cells and SPI-2 for survival/replication in phagocytic cells. However, because SPI-1 TTSS mutants are invasion-incompetent, the role of this TTSS in post-invasion processes has not been investigated. In this study, we have used two distinct methods to internalize a non-invasive SPI-1 TTSS mutant (invA) into cultured epithelial cells: (i) co-internalization with wild-type S. Typhimurium (SPI-1-dependent) and (ii) complementation with the Yersinia pseudotuberculosis invasin (inv) gene (SPI-1-independent). In both cases, internalized invA mutants were unable to replicate intracellularly, indicating that SPI-1 effectors are essential for this process and cannot be complemented by wild-type bacteria in the same cell. Analysis of the biogenesis of SCVs showed that vacuoles containing mutant bacteria displayed abnormal maturation that was dependent on the mechanism of entry. Manipulation of Salmonella-containing vacuole (SCV) biogenesis by pharmacologically perturbing membrane trafficking in the host cell increased intracellular replication of wild-type but not mutant S. Typhimurium This demonstrates a previously unknown role for SPI-1 in vacuole biogenesis and intracellular survival in non-phagocytic cells.     

Systematic analysis of the regulation of type three secreted effectors in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Background  

The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.     

Identification of a NIPSNAP homologue as host cell target for Salmonella virulence protein SpiC

Salmonella enterica uses a type III secretion system encoded by SPI-2 to target specific virulence factors into the host cytosol of macrophages to inhibit the phagosomal-lysosomal maturation pathway. This ensures survival of Salmonella inside its intracellular niche, the Salmonella -containing vacuole (SCV). One such virulence factor is SpiC, which was previously shown to interfere with intracellular vesicular trafficking. In this study we have used a yeast two-hybrid assay to identify a NIPSNAP homologue as host cell target for SpiC that we have termed TassC. In vitro and in vivo co-purification of SpiC and TassC confirm the specificity of this interaction. Suppression of TassC production compensates a SpiC production deficit and allows spiC ^– Salmonella to survive within macrophages at levels comparable to wild-type Salmonella . We hypothesize that TassC is a host cell factor that determines vesicular trafficking in macrophages and is inactivated by Salmonella SpiC.     

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1

Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.     

HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium invades intestinal epithelial cells using a type three secretion system (TTSS) encoded on Salmonella Pathogenicity Island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. We previously identified RtsA, an AraC-like protein similar to the known HilC and HilD regulatory proteins. Like HilC and HilD, RtsA activates expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 TTSS structural genes. Here we present evidence that hilA expression, and hence the SPI1 TTSS, is controlled by a feedforward regulatory loop. We demonstrate that HilC, HilD and RtsA are each capable of independently inducing expression of the hilC, hilD and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, we show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through HilD, thus inducing hilC, rtsA and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, we show that 'SPI1 inducing conditions' cause independent activation of the rtsA, hilC and hilD genes in the absence of known regulators. Our model of SPI1 regulation provides a framework for future studies aimed at understanding this complicated regulatory network.     

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.     

Formation of a novel surface structure encoded by Salmonella Pathogenicity Island 2

The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2) is essential for virulence and intracellular proliferation of Salmonella enterica. We have previously identified SPI2-encoded proteins that are secreted and function as a translocon for the injection of effector proteins. Here, we describe the formation of a novel SPI2-dependent appendage structure in vitro as well as on the surface of bacteria that reside inside a vacuole of infected host cells. In contrast to the T3SS of other pathogens, the translocon encoded by SPI2 is only present singly or in few copies at one pole of the bacterial cell. Under in vitro conditions, appendages are composed of a filamentous needle-like structure with a diameter of 10 nm that was sheathed with secreted protein. The formation of the appendage in vitro is dependent on acidic media conditions. We analyzed SPI2-encoded appendages in infected cells and observed that acidic vacuolar pH was not required for induction of SPI2 gene expression, but was essential for the assembly of these structures and their function as translocon for delivery of effector proteins.     

Salmonella type III secretion-associated protein InvE controls translocation of effector proteins into host cells

Salmonella enterica encodes a type III secretion system (TTSS) within a pathogenicity island located at centisome 63 (SPI-1), which is essential for its pathogenicity. This system mediates the transfer of a battery of bacterial proteins into the host cell with the capacity to modulate cellular functions. The transfer process is dependent on the function of protein translocases SipB, SipC, and SipD. We report here that Salmonella protein InvE, which is also encoded within SPI-1, is essential for the translocation of bacterial proteins into host cells. An S. enterica serovar Typhimurium mutant carrying a loss-of-function mutation in invE shows reduced secretion of SipB, SipC, and SipD while exhibiting increased secretion of other TTSS effector proteins. We also demonstrate that InvE interacts with a protein complex formed by SipB, SipC, and their cognate chaperone, SicA. We propose that InvE controls protein translocation by regulating the function of the Sip protein translocases.     

Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.     

Intracellular activities of Salmonella enterica in murine dendritic cells

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.